Project Summary/Abstract The organization of proteins, both in space and time, is ubiquitous in natural processes. Engineered protein systems, on the other hand, typically lack this organization. Organizing biosynthetic and regulatory systems promises to improve yields of difficult-to-produce biomolecules and provide insight into the mechanisms of organization-related pathologies. These include many neurodegenerative diseases; spatiotemporal regulation is also implicated in processes of signaling, metabolism, and aging. Self-assembling, heritable proteins, known as prions, offer a promising method to organize biochemical processes in yeast and other organisms, and in turn to reveal principles underlying organization-related dysfunction. The aims of the proposed research are three-fold: (I) Develop a toolkit of orthogonal prion domains to organize biochemistry; (II) Use prion domains to enhance biosynthesis and organize information flow; and (III) Use systems-level models to identify candidate pathways for prion-based organization. A recently discovered suite of prion-like proteins will be mined systematically for self-assembling domains conferring desirable phenotypes for biosynthetic and regulatory organization. Well-studied and newly discovered prion domains will be used to organize model enzymatic pathways and transcription factors, and to understand the effects of prion-based organization. Opioid synthesis will also be organized in this manner in order to improve titers and yields. Finally, a spatially resolved reaction-diffusion model will be implemented to examine the function of prion-based organization in detail and predict pathways that will benefit from organization in an intracellular prion phase. This model will make quantitative predictions of experimentally inaccessible quantities, such as the detailed concentration gradients within prion-based membraneless organelles. Engineering the spatiotemporal organization of biosynthetic processes has the potential to greatly increase our ability to generate useful quantities of recalcitrant biomolecules. These could range from opioid molecules and their precursors to complex macrocyclic antimicrobial compounds, all of which resist chemical synthesis and lack high-yielding biosynthetic routes. Understanding the mechanisms and function of prion-based organization also promises to reveal strategies to combat human pathologies, a growing number of which are found to be related to aberrant protein organization.